Method for isolating hepatitis a virus or spring viremia of carp virus

ABSTRACT

A method for isolating Hepatitis A virus or Spring viremia of Carp virus. A virus probe is prepared by linking a magnetic bead-conjugated Protein G with an anti-HAV (Hepatitis A Virus) antibody or an anti-rhabdovirus antibody. The virus probe is contacted with a sample to be analyzed to form a virus probe-virus complex. The virus probe-virus complex is then isolated. It may specifically isolate Hepatitis A virus or Spring viremia of Carp virus from a sample mixed viruses.

CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY

This patent application claims benefit under 35 U.S.C. 119(e), 120, 121,or 365(c), and is a National Stage entry from International ApplicationNo. PCT/KR2011/004015, filed on Jun. 1, 2011, which claims priority toKorean Patent Application No. 10-2010-0051892, filed Jun. 1, 2010,entire contents of which are incorporated herein by reference.

BACKGROUND

1. Field of the Invention

a. The present invention relates to a method for isolating Hepatitis Avirus or Spring viremia of Carp virus.

2. Description of the Related Art

Incidents due to health harm factor of foods derived from agriculturalfisheries livestock products seriously affect the health of the peopleand an economic loss of food poisoning cases caused by absences ofappropriate early detection methods and systems is 1.3 trillion won fora year.

Even if virus food poisoning is accounted by 34-40% of the entire foodpoisoning, loss caused by virus food poisoning is 400 billion won.Therefore, virus diagnostics development research which is available toprevention of virus food poisoning from local agricultural fisherieslivestock products and safe food production for consumers and producers,is a method to preventable these social losses in advance.

Although sanitary conditions of modern urban environment have been veryimproved, it has been reported that antibody retention rate forHepatitis A virus (HAV) is lowered and that is result to viral hepatitiscases is continuing to decline, whereby virus infection cases iscontinuing to increase.

Cases of Hepatitis A virus (HAV) detection in specimen from agriculturalproducts including fruits and vegetables have been reported in internaland external. However, the developed diagnosis methods for Hepatitis areA virus (HAV) have been limited in developments of the test methodsthrough mainly enzyme immunoassay using monoclonal or polyclonalantibodies and biological tests so that they are unsuitable for thefinal user in the field to use and only carried out in parts ofgroundwater, seafood, meat products and vegetables. In addition, mainviral diseases of carp and olive flounder (Paralichthys olivaceus) areviral hemorrhagic septicemia (VHS) and spring viremia of carp (SVC). TheSVC is a disease occurred in the period increasing temperature from 7°C. to 14° C. It is becoming the global issue because of its strongtoxicity and infectiousness. Although SVCV virus which is cause of theSVC has been detected in cultured carp in Korea, there is no extensivedamage caused by the SVC so far. Because it is anticipated that thedamage due to the SVC should be increased in future, appropriatemeasures are urgently needed. However, distinct therapeutic measures forviral diseases have been not established yet. In addition, in the caseof survive fishes suffered from virus diseases, viruses are remained intheir body so that they may be acted as virus carrier which may beconsistently troubled. Therefore, it is anticipated that the spread anddamage of the virus diseases should be increased. Accordingly, earlydiagnoses through understanding of the infection status of the virus inaquafarm are urgently needed on obtaining food sources.

Virus purification methods and molecular diagnostics for agriculturalfisheries livestock products currently in use have limitations asfollows:

First, typical virus purification was a manner that viruses areinoculated into the host cell and cultured with passage to dilute otherspecies such that plenty of the target species are cultured to isolate.However, such the manner was costly and time-consuming.

Second, it is possible to misjudge to non-contamination although virusesare actually present, since the various substances are present in foodand parts of them act as interfering substance to reaction of molecularspecific-gene amplification.

Throughout this application, several patents and publications arereferenced and citations are provided in parentheses. The disclosure ofthese patents and publications is incorporated into this application inorder to more fully describe this invention and the state of the art towhich this invention pertains.

SUMMARY

The present inventors have made intensive studies to develop a methodfor detecting Hepatitis A virus or Spring viremia of Carp virus from asample mixed viruses in quick and accurate manner. As a result, thepresent inventors have designed an immuno-probe by linking a magneticbead with an antibody and found a method for isolating in quick andaccurate manner.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

In one aspect of the present invention, there is provided a method forisolating Hepatitis A virus or Spring viremia of Carp virus, including:

(a) preparing a virus probe by linking a magnetic bead-conjugatedProtein G with an anti-HAV (Hepatitis A Virus) antibody or ananti-rhabdovirus antibody;

(b) contacting a sample to be analyzed with the virus probe to form avirus probe-virus complex; and

(c) isolating the virus probe-virus complex.

The step (b) may be performed at room temperature for 1-30 min.

The step (c) may be performed by contacting a magnet to the resultant ofthe step (b) to isolate the virus probe-virus complex.

The method may further include genetically analyzing the virus of thevirus probe-virus complex isolated in the step (c) by a geneamplification.

The gene amplification may be carried out using a primer pair as setforth in SEQ ID NO:1 and SEQ ID NO:2 or a primer pair as set forth inSEQ ID NO:3 and SEQ ID NO:4 to detect Hepatitis A virus.

The gene amplification may be carried out using the primer pair as setforth in SEQ ID NO:1 and SEQ ID NO:2 or the primer pair as set forth inSEQ ID NO:3 and SEQ ID NO:4 by a nested polymerase chain reaction.

The gene amplification may be carried out using a primer pair as setforth in SEQ ID NO:5 and SEQ ID NO:6 to detect Spring viremia of Carpvirus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically represents the series of processes of thepreparation for the virus probes and the detection for Hepatitis Avirus.

FIG. 2 represents results that Hepatitis A virus isolated from virusprobe-virus complex was eluted and verified using Nested PCR.

FIG. 3 schematically represents the series of processes of thepreparation for the virus probes and the detection for Spring viremia ofCarp virus.

FIG. 4 represents results that Spring viremia of Carp virus isolatedfrom virus probe-virus complex was eluted and verified using RT-PCR.

DETAILED DESCRIPTION

The present inventors have made intensive studies to develop a methodfor detecting Hepatitis A virus or Spring viremia of Carp virus from asample mixed viruses in quick and accurate manner. As a result, thepresent inventors have designed an immuno-probe by linking a magneticbead with an antibody and found a method for isolating in quick andaccurate manner.

The method for isolating Hepatitis A virus or Spring viremia of Carpvirus in the present invention is explained in detail according to thestep as follows:

Step (a): Preparation of a Virus Probe

According to the present invention, a virus probe is prepared by linkinga magnetic bead-conjugated Protein G with an anti-HAV (Hepatitis AVirus) antibody or an anti-rhabdovirus antibody.

The anti-HAV (Hepatitis A Virus) antibody or an anti-rhabdovirusantibody are prepared by various methods.

The antibody production may be prepared by a method widely known in theart, such as a fusion method (Kohler and Milstein, European Journal ofImmunology, 6:511-519 (1976)), a recombinant DNA methods (U.S. Pat. No.4,816,56) or a phage antibody library technique (Clackson et al, Nature,352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597(1991)). General process for antibody production is described in Harlow,E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold SpringHarbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manualof Techniques, CRC Press, Inc., Boca Raton, Fla., 1984; Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991, and the teachings ofwhich are incorporated herein by reference in its entity. For example,the preparation of the hybridoma cells producing monoclonal antibody isaccomplished by fusing immortalized cell line with antibody-producinglymphocytes. This can be done by techniques well known in the art.Polyclonal antibodies may be prepared by injection of the antigendescribed above to suitable animal, collecting antiserum containingantibodies from the animal, and isolating specific antibodies by any ofthe known affinity techniques.

In the present invention, the term used herein “antibody” in conjunctionwith Hepatitis A virus or Spring viremia of Carp virus refers to anantibody which is capable of specifically binding Hepatitis A virus orSpring viremia of Carp virus. The “antibody” binds specifically to thesurface protein of Hepatitis A virus or Spring viremia of Carp virus,and includes any antibody fragments as well as the entire antibody.

The entire antibody includes two full-length light chains and twofull-length heavy chains, and each light chain is linked to the heavychain by disulfide bond. The heavy chain constant region includes fivedifferent isotypes (y, μ, α, δ and ε) of which the subclass isclassified into γ1, γ2, γ3, γ4, α1 and α2. The light chain constantregion includes two different isotypes (K and λ) (Cellular and MolecularImmunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B.Saunders Co. Philadelphia, Pa. (1991); Nisonoff, A., Introduction toMolecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates,Inc., Sunderland, Mass. (1984)).

Antigen binding fragment refers to any antibody fragment capable ofbinding antigen including Fab, F(ab′), F(ab′)₂ and Fv. Fab has oneantigen binding site which is composed of one variable domain from eachheavy and light chain of the antibody, one constant region of lightchain and the first constant region (C_(H1)) of heavy chain. Fab′ isdifferent to Fab in the senses that there is a hinge region containingone or more cysteine residues at C-terminal of C_(H1) domain of heavychain. F(ab′)₂ antibody is produced by forming a disulfide bond betweencysteine residues of hinge region of Fab′. Fv is a minimal antibodyfragment including one variable region from each heavy and light chainand recombinant technique to prepare a Fv fragment is disclosed in PCTWO 88/10649, PCT WO 88/106630, PCT WO 88/07085, PCT WO 88/07086 and PCTWO 88/09344. Two-chain Fv is linked by non-covalent bond between onevariable region of each heavy and light chain, and single-chain Fv isgenerally linked by covalent bond via a peptide linker between onevariable region of each heavy and light chain or is directly linked toeach other at C-terminal, forming a dimer such as two-chain Fv. Suchantibody fragments may be obtained using a proteolytic enzymes (e.g., awhole antibody is digested with papain to produce Fab fragments, andpepsin treatment results in the production of F(ab′)₂ fragments), andmay be preferably prepared by genetic recombination techniques.

Step (b): Formation of Virus Probe-Virus Complex

Afterwards, the virus probe of step (a) is contacted with a sample to beanalyzed to form a virus probe-virus complex.

The term used herein “sample” includes any sample which may be containeda virus. Examples of the sample include animal cells, tissues, blood,plasma, agricultural fisheries livestock products and foods, but are notlimited to.

Preferably, the step (b) is performed at room temperature for 1-30 min.

According to an embodiment, the anti-HAV (Hepatitis A Virus) antibodybound to the magnetic bead-conjugated Protein G of the present inventionwas bound to the putative VP1/P2A connected proteins of HAV surface andthe anti-rhabdovirus antibody bound to the magnetic bead-conjugatedProtein G of the present invention was bound to the G protein of Springviremia of Carp virus surface.

Step (c): Isolation of Virus Probe-Virus Complex

Finally, the virus probe-virus complex is isolated from the resultant ofthe step (b).

Preferably, the step (c) is performed by contacting a magnet to theresultant of the step (b) to isolate the virus probe-virus complex.

According to an embodiment, the virus probe-virus complex isolated fromthe step (c) is eluted, neutralized to pH 7.5 and verified the virusisolation.

Preferably, the method further includes the step of geneticallyanalyzing the virus of the virus probe-virus complex isolated in thestep (c) by a gene amplification.

Where the present method is carried out the gene amplification, the geneamplification is executed by PCR using primers.

The term used herein “amplification” refers to reactions for amplifyingnucleic acid molecules. A multitude of amplification reactions have beensuggested in the art, including polymerase chain reaction (hereinafterreferred to as PCR) (U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159), reverse transcription-polymerase chain reaction (hereinafterreferred to as RT-PCR) (Sambrook, J. et al., Molecular Cloning. ALaboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), the methodsof Miller, H. I. (WO 89/06700) and Davey, C. et al. (EP 329,822), ligasechain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP439,182), transcription-mediated amplification (TMA; WO 88/10315), selfsustained sequence replication (WO 90/06995), selective amplification oftarget polynucleotide sequences (U.S. Pat. No. 6,410,276), consensussequence primed polymerase chain reaction (CP-PCR; U.S. Pat. No.4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR; U.S.Pat. Nos. 5,413,909 and 5,861,245), nucleic acid sequence basedamplification (NASBA; U.S. Pat. Nos. 5,130,238, 5,409,818, 5,554,517 and6,063,603), strand displacement amplification and loop-mediatedisothermal amplification (LAMP), but not limited to. Other amplificationmethods that may be used are described in U.S. Pat. Nos. 5,242,794,5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317.

PCR is one of the most predominant processes for nucleic acidamplification and a number of its variations and applications have beendeveloped. For example, for improving PCR specificity or sensitivity,touchdown PCR, hot start PCR, nested PCR and booster PCR have beendeveloped with modifying traditional PCR procedures. In addition,real-time PCR, differential display PCR (DD-PCR), rapid amplification ofcDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction(IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR)have been suggested for certain applications. The details of PCR can befound in McPherson, M. J., and Moller, S. G. PCR. BIOS ScientificPublishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), theteachings of which are incorporated herein by reference in its entity.

Where the present method is carried out using primers, the geneamplification is executed to analyze the nucleotide sequence of thepresent biomarkers. Because the present invention is intended to detectthe nucleotide sequence of the present biomarkers, the nucleotidesequence of the present biomarkers in samples to be analyzed (e.g.,genomic DNA) is searched to determine MS or DM.

According to an embodiment, the amplification reactions are performed byPCR (polymerase chain reaction) disclosed in U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159.

To verify the isolation of Hepatitis A virus according to the presentinvention, preferably, the gene amplification is carried out using aprimer pair as set forth in SEQ ID NO:1 and SEQ ID NO:2 or a primer pairas set forth in SEQ ID NO:3 and SEQ ID NO:4 to detect Hepatitis A virusand more preferably the gene amplification is carried out by a nestedpolymerase chain reaction.

To verify the isolation of Spring viremia of Carp virus according to thepresent invention, preferably, the gene amplification is carried outusing a primer pair as set forth in SEQ ID NO:5 and SEQ ID NO:6 todetect Spring viremia of Carp virus.

The features and advantages of the present invention will be summarizedas follows:

(a) The present invention provides a method for isolating Hepatitis Avirus or Spring viremia of Carp virus in quick and accurate manner.

(b) The present invention may specifically isolate Hepatitis A virus orSpring viremia of Carp virus from a sample mixed viruses.

(c) The present invention may detect a small quantity of viruses usingvirus-specific antigen-antibody reaction.

EXAMPLES OF THE INVENTION

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

Examples Examples 1 Immunoprecipitation Method Using Magnetic Beads

Virus probes of the present invention were prepared by linking magneticbead-conjugated Protein G as magnetic substance with an anti-HAV(Hepatitis A Virus) antibody using virus diagnostic technique. Themanufacturing process of virus probes is as follows: 50 μl of magneticbead-conjugated Protein G (Invitrogen, USA) was added in 1.5 mL tube andthe tube was attached on magnet to remove the supernatant. 10 μg ofanti-HAV antibody (Goat anti-HAV HM175 polyclonal antibody, catalog#PAB13966, putative VP1/P2A connected proteins antigen peptide,ESMMSRIAAGDLESSVDDPRSEEDKRFESHIECRKPYKELRLEVGKQRLKYAQEEL, Abnova) wasdissolved in 200 μl of PBS (Phosphate Buffered Saline) containing 0.02%Tween 20 and add into the tube mentioned above to react for 10 min.After completion of the reaction, the tube was attached on magnet toremove the supernatant such that only virus probes linked magneticbead-conjugated Protein G and antibody were remained. To removeimpurities except for virus probes, the resultant was washed with PBStwice and removed the supernatant to prepare virus probes for virusdetection.

The reaction condition of HAV and HEV (Hepatitis E Virus) mixed withvirus proves is as follows: 1 mL of HAV and HEV was added in the tubecontaining the prepared virus probes, mixed and reacted at roomtemperature for 10 min. After completion of the immunoreaction, the tubewas attached on magnet to isolate an antigen-antibody complex in whichthe antibody bound to the magnetic bead-conjugated Protein G was boundto the antigen (virus). The magnetic bead portion is bound to magnet.The supernatant containing non-binding viruses was transferred to a newtube (1.5 mL) for a PCR analysis. 200 μl of the antigen-antibody complexbinding Protein G was washed with PBS three times to remove impurities,suspended with 100 μl of PBS and moved to new 1.5 mL tube to removesupernatant. The tube containing the antigen-antibody complex bindingProtein G was eluted using eluent (50 mM glycine, pH 2.8) to isolate andthe resultant was neutralized with 100 mM Tris solution to pH 7.5 andsubjected to Nested RT-PCR to verify purification of HVA (FIG. 1).

Virus probes of the present invention were prepared by linking magneticbead-conjugated Protein G as magnetic substance with an anti-rhabdovirusantibody using virus diagnostic technique. The manufacturing process ofvirus probes is as follows: 50 μl of magnetic bead-conjugated Protein G(Invitrogen, USA) was added in 1.5 mL tube and the tube was attached onmagnet to remove the supernatant. 10 μg of anti-rhabdovirus antibody(Anti-rhabdovirus antibody was prepared using inoculating purified virusto mouse to obtain polyclonal antibody. The anti-rhabdovirus antibodywas provided from Professor Myung Ju Oh, Department of AqualifeMedicine, Chonnam National University) was dissolved in 200 μl of PBS(Phosphate Buffered Saline) containing 0.02% Tween 20 and add into thetube mentioned above to react for 10 min. After completion of thereaction, the tube was attached on magnet to remove the supernatant suchthat only virus probes including magnetic bead-conjugated Protein G andantibody were remained. To remove impurities except for virus probes,the resultant was washed with PBS twice and removed the supernatant toprepare virus probes for virus detection.

The reaction condition of VHSV (Viral Hemorrhagic Septicemia Virus) andSVCV (Spring Viraemia of Carp Virus) mixed with virus proves is asfollows: 1 mL of VHSV and SVCV was added in the tube containing theprepared virus probes, mixed and reacted at room temperature for 10 min.After completion of the immunoreaction, the tube was attached on magnetto isolate an antigen-antibody complex in which the antibody bound tothe magnetic bead-conjugated Protein G was bound to the antigen (virus).The magnetic bead portion is bound to magnet. The supernatant containingnon-binding viruses was transferred to a new tube (1.5 mL) for a PCRanalysis. 200 μl of the antigen-antibody complex binding Protein G waswashed with PBS three times to remove impurities, suspended with 100 μlof PBS and moved to new 1.5 mL tube to remove supernatant. The tubecontaining the antigen-antibody complex binding Protein G was elutedusing eluent (50 mM glycine, pH 2.8) to isolate and the resultant wasneutralized with 100 mM Tris solution to pH 7.5 and subjected to NestedRT-PCR to verify purification of SVCV (FIG. 3).

Examples 2 Verification of Hepatitis A Virus Purification

To verify purification of target virus from virus mixture withimmunoprecipitation using magnetic beads, the eluted resultant wassubjected to Nested RT-PCR to detect virus. To identify virus, PCR wasperformed using primer specific for HAV and HEV. The sequences of theprimer used in the identification are as follow: HAV_(—)2949F (5′-TATTTG TCT GTC ACA GAA CAA TCA G-3′) (SEQ ID NO: 1) and HAV_(—)3192R(5′-AGG AGG TGG AAG CAC TTC ATT TGA-3′) (SEQ ID NO: 2) were used foridentification of HAV. HEV_EX_F (5′-CAT GGT AAA GTG GGT CAG GGT AT-3′)(SEQ ID NO: 7) and HEV_EX_R (5′-AGG GTG CCG GGC TCG CCG GA-3′) (SEQ IDNO: 8) were used for identification of HEV. HAV_dkA24_F (5′-CTT CCT GAGCAT ACT TGA GTC-3′) (SEQ ID NO: 3), HAV_dkA25_R (5′-CCA GAG CTC CAT TGAACT C-3′) (SEQ ID NO: 4), HEV_IN_F (5′-GTA TTT CGG CCT GGA GTA AGA C-3′)(SEQ ID NO: 9) and HEV_IN_R (5′TCA CCG GAG TGY TTC TTC CAG AA-3′) (SEQID NO: 10) were used for Nested RT-PCR.

PCR condition for virus verification is as follows: PCR reaction mixturefor HAV was denatured for 30 sec at 94° C., subjected to 40 cycles of 1min at 60° C., 1 min at 72° C. and extended finally for 10 min at 72° C.Nested RT-PCR reaction mixture for HAV was denatured for 30 sec at 94°C., subjected to 40 cycles of 1 min at 50° C., 1 min at 72° C. andextended finally for 10 min at 72° C. PCR reaction mixture for HEV wasdenatured for 30 sec at 94° C., subjected to 40 cycles of 1 min at 61°C., 30 sec at 72° C. and extended finally for 10 min at 72° C. NestedRT-PCR reaction mixture for HEV was denatured for 30 sec at 94° C.,subjected to 35 cycles of 1 min at 61° C., 30 sec at 72° C. and extendedfinally for 10 min at 72° C. As a result, it was shown that HEVnon-bound to anti-HAV antibody and some HAV were removed and HAV boundto anti-HAV antibody was isolated by eluent solution in anantibody-specific manner (FIG. 2).

Examples 2 Verification of Spring Viremia of Carp Virus (SVCV)Purification

To verify purification of target virus from virus mixture withimmunoprecipitation using magnetic beads, the eluted resultant wassubjected to Nested RT-PCR to detect virus. To identify virus, PCR wasperformed using primer specific for SVCV and VHSV. The sequences of theprimer used in the identification are as follow: SVCV_F1 (5′-TCT TGG AGCCAA ATA GCT CAR RTC G-3′) (SEQ ID NO: 5) and SVCV_R4 (5′-CTG GGG TTT CCNCCT CAA AGY TGY-3′) (SEQ ID NO: 6) were used for identification of SVCV.VHSV_F (5′-CAG GTC CTG GAA GCA GGA AAA A-3′) (SEQ ID NO: 11) and VHSV_R(5′-CCC AGA ATG ACC CCG AAT AGG-3′) (SEQ ID NO: 12) were used foridentification of VHSV. HAV_dkA24_F (5′-CTT CCT GAG CAT ACT TGA GTC-3′)(SEQ ID NO: 3), HAV_dkA25_R (5′-CCA GAG CTC CAT TGA ACT C-3′) (SEQ IDNO: 4), HEV_IN_F (5′-GTA TTT CGG CCT GGA GTA AGA C-3′) (SEQ ID NO: 9)and HEV_IN_R (5′TCA CCG GAG TGY TTC TTC CAG AA-3′) (SEQ ID NO: 10) wereused for Nested RT-PCR.

PCR conditions for SVCV and VHSV detection are as follows: PCR reactionmixture for SVCV was denatured for 1 min at 95° C., subjected to 30cycles of 1 min at 55° C., 1 min at 72° C. and extended finally for 10min at 72° C. PCR reaction mixture for VHSV was denatured for 1 min at95° C., subjected to 30 cycles of 1 min at 58° C., 1 min at 72° C. andextended finally for 5 min at 72° C. As a result, the SVCV band wasobserved to show that SVCV was isolated (FIG. 4).

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

1. A method for isolating Hepatitis A virus or Spring viremia of Carpvirus, comprising: (a) preparing a virus probe by linking a magneticbead-conjugated Protein G with an anti-HAV (Hepatitis A Virus) antibodyor an anti-rhabdovirus antibody; (b) contacting a sample to be analyzedwith the virus probe to form a virus probe-virus complex; and (c)isolating the virus probe-virus complex.
 2. The method according toclaim 1, wherein the step (b) is performed at room temperature for 1-30min.
 3. The method according to claim 1, wherein the step (c) isperformed by contacting a magnet to the resultant of the step (b) toisolate the virus probe-virus complex.
 4. The method according to claim1, further comprising genetically analyzing the virus of the virusprobe-virus complex isolated in the step (c) by a gene amplification. 5.The method according to claim 4, wherein the gene amplification iscarried out using a primer pair as set forth in SEQ ID NO:1 and SEQ IDNO:2 or a primer pair as set forth in SEQ ID NO:3 and SEQ ID NO:4 todetect Hepatitis A virus.
 6. The method according to claim 5, whereinthe gene amplification is carried out using the primer pair as set forthin SEQ ID NO:1 and SEQ ID NO:2 or the primer pair as set forth in SEQ IDNO:3 and SEQ ID NO:4 by a nested polymerase chain reaction.
 7. Themethod according to claim 4, wherein the gene amplification is carriedout using a primer pair as set forth in SEQ ID NO:5 and SEQ ID NO:6 todetect Spring viremia of Carp virus.